Cell cultures are instrumental for studying basic and applied aspects of gene therapy for cytic fibrosis (CF). Human airway epithelial cells maintained at an air-liquid interface display mucociliary differentiation similar to the in vivo epithelium. These cells faithfully reproduce the high resistance to vectors, characteristic of the native mucosa, providing a strong platform for pre-clinical studies to address issues crucial to the success of gene therapy for CF. A Tissue Procurement and Cell Culture Core was established at the University of North Carolina (UNC) in 1984, under the auspices of the CF Foundation, to provide standardized cell cultures to CF researchers. The Core has supported UNC Gene Therapy for CF projects since 1993 and has increased its output and capabilities to meet growing research demands. The Core routinely makes available cells and media that are unavailable and/or prohibitively expensive if purchased commercially. The present application will provide continuity in the supply of human airway epithelial cell cultures to UNC Gene Therapy for Cystic Fibrosis Program Project investigators by providing the following specific functions: 1) Obtain airway tissues from normal, CF and disease control humans as the source for primary cells, for acute ex vivo experiments, protein and RNA isolation, immunostaining and in situ hybridization. Characterize donors while protecting confidentiality. 2) Isolate primary epithelial cells from excised human airways. Prepare and maintain primary epithelial cell cultures. Provide support for preparation of well differentiated airway epithelial cultures from passaged or cryopreserved primary human cells. Optimize culture conditions to replicate gene expression, morphological differentiation, and physiologic functions of airways as evidenced by rotational mucus transport and regulation of the airway surface liquid (ASL) layer. Through these functions, and in conjunction with other Cores in this application, the Cell Culture Core will support research towards the development of safe and effective gene therapy for CF.